Mammalian spermatozoa are vulnerable to membrane damage resulting in motility loss due to spontaneous peroxidation of phospholipids by O2, which places an ultimate limit on their motile lifetime in the oviduct. Previous work carried out with rabbit and mouse sperm had shown that the rate was linear in PO2 and had EA=78kcal/mol. Work in this grant period showed that peroxidation in human sperm had identical properties, but that the rate as reflected in time to motility loss was highly variable and correlated strongly with superoxide dismutase (SOD) activity. Inactivation of human sperm glutathione peroxidase (GPX) drastically shortens time to motility loss. These results imply that lipid hydroperoxide ROOH and superoxide radical (SO) play a key role in peroxidation. A second finding was that intact human sperm incorporate radiolabelled exogenous fatty acids, both saturated and unsaturated. The polyunsaturated ones are lost during peroxidation and so provide a way of probing the mechanisms of lipid breakdown more closely. Incorporation of fatty acids provides a way of replacing fatty acids damaged by peroxidation and so modulating lipid peroxidation. The analytical techniques developed during this period have led to high resolution chromatographic techniques for glycolipids and so offers experimental access to peroxidative effects on sperm glycolipids. From the combination of these results have come the four specific aims for this proposal. 1) To establish the mechanism of the key reaction leading to peroxidative damage of sperm phospholipids. The hypothesis is that ROOH reacts directly with O2 to produce lipid breakdown and that ROOH production depends on SO. 2) To utilize the incorporation of radiolabelled polyunsaturated acids into the phospholipid pool of intact sperm to characterize the set of breakdown products of the particular fatty acid. The hypothesis is that each set will be unique. 3) To determine the effect of fatty acid incorporation into intact sperm under conditions of lipid peroxidation on sperm lifetime. The hypothesis is that this will increase sperm lifetime. 4) To characterize and quantitate the acidic and neutral glycolipids in mouse, rabbit, bovine and human sperm and to determine the damage to these components due to peroxidation. The hypothesis is that sperm glycolipids play an important role in plasma membrane function. These baseline data will render the hypothesis accessible to experimental test. Successful pursuit of these aims depends heavily on the use of sensitive, high resolution thin layer chromatography techniques which have been developed for this project during the past grant period. Greater emphasis will be placed on the investigation of human sperm, whose susceptibility to spontaneous peroxidation reactions may be a major component of unexplained male infertility. It is anticipated that the results from this proposed research may help to alleviate this condition.